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Single-Step Purification of Recombinant Thermus aquaticus DNA Polymerase Using DNA-Aptamer Immobilized Novel Affinity Magnetic Beads

Authors

  • Huseyin Avni Oktem,

    1. Department of Biological Science, Nanotechnology-Nanobiotechnology Research Center, Middle East Technical University, 06531 Ankara, Turkey
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  • Gulay Bayramoglu,

    1. Biochemical Processing and Biomaterial Research Laboratory, Faculty of Science, Kırıkkale University, 71450 Yahşihan, Kırıkkale, Turkey
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  • V. Cengiz Ozalp,

    1. Department of Biological Science, Nanotechnology-Nanobiotechnology Research Center, Middle East Technical University, 06531 Ankara, Turkey
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  • M. Yakup Arica

    Corresponding author
    1. Biochemical Processing and Biomaterial Research Laboratory, Faculty of Science, Kırıkkale University, 71450 Yahşihan, Kırıkkale, Turkey
    • Biochemical Processing and Biomaterial Research Laboratory, Faculty of Science, Kırıkkale University, 71450 Yahşihan, Kırıkkale, Turkey. Tel: +90–318–3572477. Fax: +90–318–357–2329
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Abstract

A DNA aptamer specific for Thermus aquaticus DNA polymerase (Taq-polymerase) was immobilized on magnetic beads, which were prepared in the presented study. The effect of various parameters including pH, temperaturem and aptamer concentration on the immobilization of 5′-thiol labeled DNA-aptamer onto glutaric dialdhyde activated magnetic beads was evaluated. The binding conditions of Taq-polymerase on the aptamer immobilized magnetic beads were studied using commercial Taq-polymerase to characterize the surface complexation reaction. Efficiency of affinity magnetic beads in the purification of recombinant Taq-polymerase from crude extracts was also evaluated. For this case, the enzyme “recombinant Taq-DNA polymerase” was cloned and expressed using an Amersham E. coli GST-Gene Fusion Expression system. Crude extracts were in contact with affinity magnetic beads for 30 min and were collected by magnetic field application. The purity of the eluted Tag-polymerase from the affinity beads, as determined by HPLC, was 93% with a recovery of 89% in a one-step purification protocol. Apparently, the system was found highly effective as one step for the low-cost purification of Taq-polymerase in bacterial crude extract.

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